PROJECT 5 - Net (PETERLIN AND ALBER. PROJECT LEADERS! Nef (negative factor) is a misnamed, accessory activator of HIV-1, HIV-2, and SIV that is essential for high-level viremia and progression to AIDS in infected patients. Nef is myristylated at its N-terminus and binds cholesterol via its C-terminal cholesterol recognition motif (CRM). These signals target Nef to lipid rafts (LRs) in cellular membranes. This project aims to characterize Nef interactions with partner proteins that are mobilized in two crucial processes[unreadable]host-cell activation and receptor endocytosis. These studies will test the hypotheses that much of the surface of Nef contacts host proteins, and these interactions not only bring together functional partners but also activate targets through allosteric conformational changes. The identification of surfaces of Nef that mediate multiple host interactions will provide new information about how to simultaneously target more than one Nef function with new Pharmaceuticals. Nef assembles a signalosome early in the viral life cycle that activates infected immune cells, stimulating viral replication. The signalosome consists of six proteins including the Nef scaffold. It is not known if Nef simply brings together the host components of the signalosome or also activates these factors through allosteric interactions. Nef also mediates remodeling of the host cell surface by promoting internalization of cellular receptors, such as CD4, MHC class I, and T cell antigen receptor. To mediate endocytosis, Nef bridges the receptors in ternary complexes with armadillo repeat (ARM-repeat) containing proteins such as adaptor protein (AP) complexes, [unreadable]COP, and the catalytic subunit (V1H) of the vacuolar ATPase (V-ATPase). The mechanisms of these essential bridging functions are unknown and the cooperativity of Nef binding has not been characterized. Monomeric and dimeric structures of Nef have been solved by NMR and crystallography, but except for small complexes between the proline-rich sequence of Nef and SH3 domains, little information is available about Nef bound to host proteins.